Method of drug formulation based on increasing the affinity of active agents for crystalline microparticle surfaces

ABSTRACT

Methods are provided for promoting the adsorption of an active agent to microparticles by modifying the structural properties of the active agent in order to facilitate favorable association to the microparticle.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/746,656 filed Jun. 22, 2015, which is a divisional of U.S.patent application Ser. No. 12/883,369 filed Sep. 16, 2010, which is acontinuation of U.S. patent application Ser. No. 11/532,065 filed Sep.14, 2006 and claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. Nos. 60/717,524 filed on Sep. 14, 2005, andNo. 60/744,882, filed on Apr. 14, 2006, the entire contents of eachwhich are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to drug formulations and is particularly relatedto methods. More specifically, binding or adsorbing active agents ontothe surface of crystalline microparticles is disclosed.

BACKGROUND OF THE INVENTION

Delivery of therapeutic agents has been a major problem. Oraladministration is one of the most common and preferred routes ofdelivery due to ease of administration, patient compliance, anddecreased cost. However, the disadvantages of this route include low orvariable potency and inefficient adsorption of the therapeutic. This isparticularly evident when the compound to be delivered is unstable underconditions encountered in the gastrointestinal tract. A variety ofcoatings and encapsulation methods have been developed in the art, butonly a few are effective in addressing this issue. Still, there aretherapeutic compounds that tend to be less active in the conditions ofthe gastrointestinal tract and must be administered in higher dosages tobe adsorbed into the bloodstream in an effective amount.

A broad range of drug formulation systems have been developed to addressthe problem of optimal drug delivery and are based on incorporation ofdrug into a matrix that acts as a carrier. Factors considered in drugformulation include requirements that the system be non-toxic andnon-reactive with the drug to be delivered, economical to manufacture,formed of readily available components, and consistent with respect tofinal composition and physical characteristics, including stability andrelease rate. It is also preferable that the drug delivery system isformed of materials easily removed from the body by normal physiologicprocesses.

Advancements in microparticle technology have aided in the developmentof improved drug formulations. However, despite these advances there isstill a need in the art for stable drug formulations having long termeffectiveness and optimal adsorption when administered as apharmaceutical, particularly by pulmonary means. One approach inaddressing this deficiency is to target the structuralcharacteristics/properties of the active agent that would promote itsadsorption to the microparticle surface and decrease its tendency toremain in solution.

SUMMARY OF THE INVENTION

Methods are provided for binding, coating or adsorbing an active agentonto a crystalline microparticle surface. In general, microparticles arecoated with an active agent by modifying the system comprising themicroparticles and the dissolved active agent such that the active agenthas a greater affinity for the microparticle surface than for remainingin solution. In particular the present invention seeks to furtherpromote the adsorption of an active agent to the microparticle surfaceby modifying/utilizing the properties of the active agent under a numberof conditions in solution.

Thus, in the present invention there is provided a method for promotingbinding of an active agent to a preformed crystalline microparticle insuspension comprising the steps of: i) modifying the chemical potentialof the active agent wherein the modifying allows for an energeticallyfavorable interaction between the active agent and microparticleindependent of removal of solvent; and ii) adsorbing the active agentonto the surface of the microparticle.

In particular embodiments of the present invention, modifying thechemical potential comprises modifying the structure, flexibility,rigidity, solubility or stability of the active agent, individually orin combination. Modifying the chemical potential of the active agentcomprises altering solution conditions. Altering solution conditionscomprises adding an active agent modifier to the solution.

In particular embodiments, the active agent modifier is selected fromthe group consisting of salts, surfactants, ions, osmolytes, alcohols,chaotropes, kosmotropes, acids, bases, and organic solvents. In oneembodiment, the salt is sodium chloride.

In still yet another embodiment of the present invention, the methodfurther comprises the step of dissolving the active agent in the fluidphase of a suspension of microparticles and changing the pH of the fluidphase. In one aspect the step of dissolving the active agent in a fluidphase refers to the dissolving of a solid. In another aspect the step ofdissolving the active agent refers to the addition of a concentratedsolution of the active agent.

In another embodiment of the present invention, the active agentmodifier improves the structural stability of the active agent.

In yet another embodiment of the present invention the active agent is aprotein, peptide, polypeptide, small molecule, or nucleic acid molecule.In another embodiment of the present invention the active agent isselected from the group consisting of insulin, ghrelin, growth hormone,and parathyroid hormone (PTH). The active agent can comprise an antibodyor antibody fragment. In various aspects of the invention the antibodycan recognize a disease-associated antigen including, withoutlimitation, a tumor-associated antigen or an infectious pathogen-relatedantigen.

In still yet another embodiment of the present invention, the smallmolecule is an ionizable molecule or a hydrophobic molecule such as, butnot limited to, cyclosporin A.

In another embodiment of the present invention, modifying the chemicalpotential of the active agent comprises modulating one or moreenergetically favorable interactions such as, but not limited to,electrostatic interactions, hydrophobic interactions, and/or hydrogenbonding interactions between the active agent and the microparticlesurface. In one embodiment, the microparticle comprises adiketopiperazine such as, but not limited to, fumaryl diketopiperazine.

In yet another embodiment of the present invention, the method furthercomprises a step for removing or exchanging the solvent. Solvent, asused herein, refers to the fluid medium in which the active agent andmicroparticle are “bathed.” It should not be interpreted to require thatall components are in solution. Indeed in many instances it may be usedto refer to the liquid medium in which the microparticles are suspended.

In another embodiment of the present invention, there is provided aprocess for preparing a drug delivery composition comprising an activeagent and a crystalline microparticle comprising the steps of: providingan active agent solution comprising an active agent molecule; modifyingthe chemical potential of the active agent; providing a microparticle ina suspension or powder; and combining the active agent solution with themicroparticle suspension or powder. The powder can be, for example,filtered but not dried.

In another embodiment of the present invention, the process of modifyingthe chemical potential of the active agent allows for interactionbetween the active agent and a microparticle. In one embodiment,modifying the chemical potential of the active agent comprises adding anactive agent modifier to the solution. Such an active agent modifier canselected from the group consisting of salts, surfactants, ions,osmolytes, alcohols, chaotropes, kosmotropes, acid, base, and organicsolvents. In yet another embodiment, the modifier decreases thesolubility of the active agent molecule, promotes association betweenthe active agent and a microparticle such as a diketopiperazineparticle, and/or improves the structural stability of the active agentmolecule.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the examplesdisclosed herein. The invention may be better understood by reference toone or more of these drawings in combination with the detaileddescription of specific embodiments presented herein.

FIGS. 1A-1C depict the effects of chaotropes and kosmotropes on loadingcurves for active agents onto fumaryl diketopiperazine (FDKP)microparticles as a function of pH and 100 mM chaotropic/kosmotropicagent according to the teachings of the present invention. FIG. 1Adepicts the loading of 0.75 mg/mL insulin onto 5 mg/mL FDKPmicroparticles in the presence of chaotropes and kosmotropes at pH3.0-5.0. FIG. 1B depicts the loading of 0.25 mg/mL glucagon-like peptide1 (GLP-1) onto 5 mg/mL FDKP microparticles in the presence of chaotropesand kosmotropes at pH 2.0-4.0. FIG. 1C depicts the loading of 0.25 mg/mLparathyroid hormone (PTH) onto 5 mg/mL FDKP microparticles in thepresence of the strong chaotropes, NaSCN and NaClO₄, between pH 4.0-5.0.

FIGS. 2A-2C depict the effects of osmolytes on loading curves for activeagents onto FDKP microparticles as a function of pH and osmolytes (100mM) according to the teachings of the present invention. FIG. 2A depictsthe loading of 0.75 mg/mL insulin onto 5 mg/mL FDKP microparticles inthe presence of osmolytes at pH 3.0-5.0. FIG. 2B depicts the loading of0.25 mg/mL GLP-1 onto 5 mg/mL FDKP microparticles in the presence ofosmolytes between pH 2.0-4.0. FIG. 2C depicts the loading of 0.10 mg/mLghrelin peptide onto 5 mg/mL FDKP microparticles in the presence ofstrong osmolytes at pH 4.0-5.0.

FIGS. 3A-3D depict the effects of alcohols on loading curves for activeagents onto FDKP microparticles as a function of pH and alcoholsaccording to the teachings of the present invention. FIG. 3A depicts theloading of 0.10 mg/mL ghrelin onto 5 mg/mL FDKP microparticles in thepresence of hexafluoroisopropanol (HFIP) at 5%, 10%, 15%, and 20% v/vbetween pH 2.0-4.0. FIG. 3B depicts the loading of 0.10 mg/mL ghrelinonto 5 mg/mL FDKP microparticles in the presence of trifluoroethanol(TFE) at 5%, 10%, 15%, and 20% v/v between pH 2.0-4.0. FIG. 3C and 3Ddepict the loading of 0.25 mg/mL GLP-1 onto 5 mg/mL FDKP microparticlesat pH 2.0-5.0 in the presence of HFIP and TFE, respectively.

FIGS. 4A-4D depict the effects of salt on loading curves for activeagents onto FDKP microparticles as a function of pH and NaClconcentration according to the teachings of the present invention. FIG.4A depicts the loading of 0.75 mg/mL insulin onto 5 mg/mL FDKPmicroparticles in the presence of 0-500 mM NaCl at pH 2.0-5.0. FIG. 4Bdepicts the loading of 0.25 mg/mL GLP-1 onto 5 mg/mL FDKP microparticlesin the presence of 0-500 mM NaCl at pH 2.0-5.0. FIG. 4C depicts theloading of 0.25 mg/mL PTH peptide onto 5 mg/mL FDKP microparticles inthe presence of 0-1000 mM NaCl at pH 2.0-5.0. FIG. 4D depicts thesecondary structural analysis of PTH at various salt concentrations (20°C.). The far-UV CD of 4.3 mg/mL PTH at pH 5.8 illustrates that as theconcentration of NaCl increases the secondary structure of the peptideadopts a more helical conformation.

FIGS. 5A-5B depict the adsorption of hydrophobic molecules ontomicroparticles according to the teachings of the present invention. FIG.5A depicts the binding of cyclosporin A to FDKP microparticles withincreasing anti-solvent (water) at 60%, 80% and 90% concentration. FIG.5B depicts the percent of theoretical maximum load achieved forcyclosporin A at varying mass ratios of cyclosporin A/FDKPmicroparticles in the presence of 90% anti-solvent.

FIG. 6 depicts the pharmacokinetics of single intravenous injection (IV)and pulmonary insufffaltion (IS) in rats using various mass ratios ofcyclosporin A/FDKP microparticles at 90% anti-solvent according to theteachings of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods useful for stabilizing pharmaceuticalactive agents in combination with crystalline microparticles. Theresulting compositions provide stable active agents coated onto thecrystalline microparticle surfaces.

The substance to be coated or adsorbed onto the crystallinemicroparticle is referred to herein as active agent. Examples of classesof active agent include pharmaceutical compositions, syntheticcompounds, and organic macromolecules that have therapeutic,prophylactic, and/or diagnostic utility.

Generally, most active agents can be coated or adsorbed onto the surfaceof crystalline microparticles including, but not limited to, organicmacromolecules, nucleic acids, synthetic organic compounds,polypeptides, peptides, proteins, polysaccharides and other sugars, andlipids. Peptides, proteins, and polypeptides are all chains of aminoacids linked by peptide bonds. Peptides are generally considered to beless than 30 amino acid residues but may include more. Proteins arepolymers that can contain more than 30 amino acid residues. The termpolypeptide as is know in the art and as used herein, can refer to apeptide, a protein, or any other chain of amino acids of any lengthcontaining multiple peptide bonds, though generally containing at least10 amino acids. The active agents used in the coating formulation canfall under a variety of biological activity classes, such as vasoactiveagents, neuroactive agents, hormones, anticoagulants, immunomodulatingagents, cytotoxic agents, antibiotics, antivirals, antigens, andantibodies.

Examples of active agents that may be employed in the present inventioninclude, in a non-limiting manner: growth hormone, antibodies andfragments thereof alkynes, cyclosporins (e.g. cyclosporin A), PPACK(D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, CMFDA(5-chloromethylfluorescein diacetate), Texas Red, clopiogrel,granulocyte macrophage colony stimulating factor (GM-CSF), glucagon-likepeptide 1 (GLP-1), ghrelin, parathyroid hormone (PTH), insulin andinsulin analogs (e.g., aspart insulin and insulin) and antibodies andfragments thereof, including, but not limited to: humanized or chimericantibodies; F(ab), F(ab)2, or single-chain antibody alone or fused toother polypeptides; therapeutic or diagnostic monoclonal antibodies tocancer antigens, cytokines, infectious agents, inflammatory mediators,hormones, and cell surface antigens. Non-limiting examples of antibodiesto tumor antigens include anti-SSX-241-49 (synovial sarcoma, Xbreakpoint 2), anti-NY-ESO-1 (esophageal tumor associated antigen),anti-PRAME (preferentially expressed antigen of melanoma), anti-PSMA(prostate-specific membrane antigen), anti-Melan-A (melanoma tumorassociated antigen), anti-tyrosinase (melanoma tumor associatedantigen), and anti-MOPC-21 (myeloma plasma-cell protein).

Microparticles

Essentially, the term “microparticle” refers to a particle with adiameter of about 0.5-1000 @m, irrespective of the precise exterior orinterior structure. Within the broad category of microparticles,“microspheres” refers to microparticles with uniform spherical shape.Crystalline microparticles as used herein refers to microparticles thathave the internal structure, though not necessarily the external form,of a crystal and have a regular arrangement of atoms in a space lattice.Ionizable crystalline surfaces refer to crystalline microparticles thathave the additional capacity to carry an electrical charge. In someembodiments the microparticle can be a single regularly shaped crystal.In various preferred embodiments the microparticle is irregularlyshaped, is porous, has dissolved active agent-accessible interiorsurfaces, or comprises multiple crystals, in any combination. Suchcharacteristics will generally increase surface area and thereby loadingcapacity. Such characteristics can also contribute to advantageousaerodynamic properties, important if the active agent is to be deliveredby inhalation of a dry powder comprising the microparticles.

Preferably, the chemical substance composing the crystallinemicroparticle is reversibly reactive with the active agent to bedelivered, non-toxic, as well as non-metabolized by rodents and humans.The foregoing notwithstanding, some levels of toxicity are tolerable,depending, for example, on the severity of the condition to be treatedor the amount of the substance to which a patient is exposed. Similarly,it is not required that the substance be completely metabolically inert.In addition, the crystalline structure of preferred microparticles isnot substantially disrupted in the process of coating or binding withactive agent. The composition of the crystalline microparticledetermines what type of chemical interactions can be manipulated todrive adsorption of an active agent to the microparticle surface.

A number of substances can be used to form crystalline microparticles.Microparticles as such have surfaces, the properties of which can bemanipulated in the coating process as disclosed in copending U.S. patentapplication Ser. No. 11/532,063 (Attorney Docket No. 51300-00025), filedon the same date as the instant application, and U.S. ProvisionalApplication Ser. No. 60/717,524 filed on Sep. 14, 2005, each of which ishereby incorporated by reference in its entirety. Representativematerials from which crystalline microparticles can be formed include,but are not limited to, aromatic amino acids, or compounds with limitedsolubility in a defined pH range such as diketopiperazines andmorpholine sulfates.

One particular example of microparticles as contemplated in the presentinvention are diketopiperazine (DKP) microparticles. As discussedherein, DKP microparticles are employed to facilitate the adsorption ofthe active agent. U.S. Pat. Nos. 5,352,461 and 5,503,852, each of whichis incorporated herein by reference in its entirety, describe a drugdelivery system based on formation of diketopiperazine (DKP)microparticles from diketopiperazine derivatives such as3,6-bis[N-fumaryl-N-(n-butyl)amino] (also referred to as fumaryldiketopiperazine or FDKP; also termed(E)-3,6-bis[4-(N-carboxy-2-propenyl)amidobutyl]-2,5-diketopiperazine)that are stable at low pH and dissolve at the pH of blood or the smallintestine. A system based on diketopiperazine structural elements or oneof its substitution derivatives, including, but not limited to,diketomorpholines and diketodioxanes, forms microparticles withdesirable size distributions and pH ranges as well as good payloadtolerance. A wide range of stable, reproducible characteristics can begenerated with appropriate manipulations of the substituent groups.These patents disclosed precipitation of the DKP in the presence of theactive agent to form microparticles comprising the active agent. Furtherdetails for synthesis, preparation, and use of diketopiperazines anddiketopiperazine microparticles are disclosed in U.S. Pat. Nos.6,071,497; 6,331,318; 6,428,771 and U.S. Patent Publication Nos.20060040953 and 20060041133, each incorporated herein by reference intheir entirety. Compositions comprising diketopiperazine particles aredisclosed in U.S. Pat. No. 6,991,779 and U.S. Patent Publication No.20040038865; each incorporated herein by reference in their entirety.

Other diketopiperazines contemplated in the present invention include3,6-di(4-aminobutyl)-2,5-diketopiperazine;3,6-di(succinyl-4-aminobutyl)-2,5-diketopiperazine (succinyldiketopiperazine or SDKP);3,6-di(maleyl-4-aminobutyl)-2,5-diketopiperazine;3,6-di(citraconyl-4-aminobutyl)-2-5-diketopiperazine;3,6-di(glutaryl-4-aminobutyl)-2,5-diketopiperazine;3,6-di(malonyl-4-aminobutyl)-2,5-diketopiperazine;3,6-di(oxalyl-4-aminobutyl)-2,5-diketopiperazine and derivativestherefrom. Diketopiperazine salts may also be utilized in the presentinvention and may included, for example, a pharmaceutically acceptablesalt such as the Na, K, Li, Mg, Ca, ammonium, or mono-, di- ortri-alkylammonium (as derived from triethylamine, butylamine,diethanolamine, triethanolamine, or pyridines, and the like). The saltmay be a mono-, di-, or mixed salt. Higher order salts are alsocontemplated for diketopiperazines in which the R groups contain morethan one acid group. In other aspects of the invention, a basic form ofthe agent may be mixed with the diketopiperazine in order to form a saltlinkage between the drug and the diketopiperazine, such that the drug isa counter cation of the diketopiperazine. DKP salts for drug deliveryare disclosed in a further detail in U.S. Patent Application PublicationNo. 20060040953 which is herein incorporated by reference in itsentirety.

U.S. Pat. Nos. 6,444,226, and 6,652,885, each herein incorporated byreference in their entirety, describe preparing and providingmicroparticles of DKP in aqueous suspension to which a solution ofactive agent is added, and then the critical step of lyophilizing thesuspension to yield microparticles having a coating of active agent. Thebasis for this formulation is that the coating of microparticle withactive agent is driven by removal of the liquid medium bylyophilization. (See also U.S. Pat. No. 6,440,463 which is incorporatedherein by reference in its entirety). In contrast to teachings in theprior art, the present invention provides means for adjusting theassociation of active agent with the microparticle prior to solventremoval. Thus, removal of the liquid medium by bulk physical methods(e.g., filtration or sedimentation) or evaporative methods (e.g.,lyophilization or spray-drying) can result in comparable loads.

Promoting Adsorption of Active Agents

Adsorbing active agent to the surface of a crystalline microparticle caninvolve altering the properties of the active agent in a solution orfluid suspension under various solution conditions, thereby promotingadsorption to the microparticle surface and reducing the amount ofactive agent remaining in solution. Alteration or modifications to theactive agent may occur with the use of modifiers such as, but notlimited to, chaotropes and kosmotropes, salts, organics such as, but notlimited to, alcohols, osmolytes, and surfactants. These modifiers canact on the active agent to alter its chemical potential and thereby itsstructure, flexibility, rigidity or stability, without chemicallyaltering the agent itself. The term “chemical potential” is well knownto one of ordinary skill. In embodiments of the present invention,“chemical potential” refers to the free energy necessary to drive achemical reaction such as, for example, interaction between an activeagent and a solvent or the adsorption of active agent onto amicroparticle. The term “energetically favorable” as used herein refersto the lowering of the free energy levels of the absorbed states of theactive agent onto the microparticle in relation to the free energy levelof uncoated microparticle, or unbound active agent and/or the insolubleforms (including aggregation or precipation) of the active agent. Theterm “structure” as used herein refers to the secondary structure of theactive agent molecule and includes the alpha-helical formation, betasheets, or random coil (unordered) of the active agent molecule, such asa protein. Additionally, the term structure may also include teritaryand quaternary structures of the molecule but is not limited to such andmay also refer to the self association, aggregation, multimerization,dimerization, and the like, of a molecule. The term “stability” as usedherein refers to the stabilization or destabilization of the structureof the active agent in the presence of the modifier.

In addition, altering the properties of the active agent in a solutionor fluid suspension are likely to affect the interactions due tohydrophobic properties, hydrogen bonding properties, and electrostaticproperties of the active agent and/or microparticle.

Hydrophobic interactions are associations of non-polar groups with eachother in aqueous solutions because of their insolubility in water.Hydrophobic interactions can affect a number of molecular processesincluding, but not limited to, structure stabilization (of singlemolecules, complexes of two or three molecules, or larger assemblies)and dynamics, and make important contributions to protein-protein andprotein-ligand binding processes. These interactions are also known toplay a role in early events of protein folding, and are involved incomplex assembly and self-assembly phenomena (e.g., formation ofmembranes).

Hydrogen bonding interactions are especially strong dipole-dipole forcesbetween molecules; a hydrogen atom in a polar bond (e.g., H—F, H—O orH—N) can experience an attractive force with a neighboringelectronegative molecule or ion, which has an unshared pair of electrons(typically an F, O, or N atom on another molecule). Hydrogen bonds areresponsible for the unique properties of water and are very important inthe organization of biological molecules, especially in influencing thestructure of proteins and DNA.

Electrostatic interactions are attractions between opposite charges orrepulsions between like charges that grow stronger as the charges comecloser to each other. Electrostatic interactions constitute a keycomponent in understanding interactions between charged bodies in ionicsolutions. For example, the stability of colloidal particles dispersedin a solvent can be explained by considering the competition betweenrepulsive electrostatic interactions and the attractive van der Waalsinteractions. Electrostatic interactions are also of importance whenconsidering interaction and adhesion between particles.

Salts

In some embodiments of the present invention, the properties of theactive agent are altered using a salt such as, but not limited to,sodium chloride. Active agents, for example, PTH and GLP-1, undergonoticeable structural changes in the presence of salt. As shown inExample 5 (FIG. 4D), the presence of salt increases the secondarystructure of PTH by promoting a more helical conformation of thepeptide. Salt has also been shown to affect the structure of GLP-1, asdisclosed in U. S. Provisional Patent Application Ser. No. 60/744,882,filed on Apr. 14, 2006 and incorportated herein by reference in itsentirety. Furthermore, salts and other ionic compounds are capable ofeither stabilizing or destabilizing proteins and peptides, especiallywhen the difference between the pH of the solution and the pI of theprotein or peptide becomes greater, by binding to specifically chargedresidues (Antosiewiez J, et al., J. Mol. Biol. 238:415-436, 1994).

Chaotropes

Chaotropes, as are well known in the art, are ions that exhibit weakinteractions with water and therefore destabilize molecules such asproteins or peptides. These compounds break down the hydrogen-bondednetwork of water and decrease its surface tension, thus promoting morestructural freedom and denaturation of proteins and peptides. Examplesof chaotropes include, but are not limited to, NaSCN, (CH₃)₃N-HCl,Na₂NO₃, and NaClO₄ and cesium chloride (CsCl).

Kosmotropes or lyotropes, on the other hand, are ions that displaystrong interactions with water and generally stabilize macromoleculessuch as proteins and peptides. This stabilization effect is broughtabout by increasing the order of water and increasing its surfacetension. Examples of kosmotropes include, but are not limited to, sodiumcitrate (Na Citrate), and sodium sulfate (Na₂SO₄).

Alcohols

Another class of modifier of active agent employed in the presentinvention is alcohols. Alcohols are able to disrupt the native structureof proteins and peptides and are also able to stabilize and induce6-helical conformations in macromolecules, most notably withinunstructured proteins and polypeptides. Such alcohols may include, butare not limited to, methanol (MeOH), ethanol (EtOH), trifluoroethanol(TFE), and hexafluoroisopropanol (HFIP). Of those, TFE and HFIP are twoof the most potent alcohols for inducing helical transitions in peptidesand proteins (Hirota et al., Protein Sci., 6:416-421; 1997, incorporatedherein by reference for all it contains regarding helical transitions inpeptides and proteins). These alcohols may affect the structure ofproteins and peptides through their ability to disrupt thehydrogen-bonding properties of the solvent (see Eggers and Valentine,Protein Sci., 10:250-261; 2001, incorporated herein by reference for allit contains regarding the effect of alcohols on the structure ofproteins).

Osmolytes

Another class of modifier that affects the active agent affinity for themicroparticle is osmolytes. Osmolytes, as are well known to the skilledartisan, are small compounds that are produced by the cells of mostorganisms in high stress situations (such as extreme temperaturefluctuations, high salt environments, etc.) to stabilize theirmacromolecules. They do not interact with the macromolecule directly butact by altering the solvent properties in the cellular environment andso their presence indirectly modifies the stability of proteins. Thesecompounds include various polyols, sugars, polysaccharides, organicsolvents, and various amino acids and their derivatives. Although themechanism of osmolytes are yet to be elucidated, it is speculated thatthese compounds likely act by raising the chemical potential of thedenatured state relative to the native state, thereby increasing the(positive) Gibbs energy difference (AG) between the native and denaturedensembles (Arakawa and Timasheff, Biochemistry 29:1914-1923;1990).

Osmolytes as contemplated in the present invention, include in anon-limiting manner, hexylene-glycol (Hex-Gly), trehalose, glycine,polyethylene glycol (PEG), trimethylamine N-oxide (TMAO), mannitol, andproline.

General Description of the Method

In the methods of the present invention, at least three components arecombined in a liquid medium: at least one active agent, (preformed)microparticles, and at least one active agent modifier as describedabove. The components of this system may be combined in any order. Insome embodiments the modifier and active agent are combined with eachother prior to that mixture being combined with a suspension ofmicroparticles. In other embodiments the agent and microparticles arefirst combined and then the modifier is added. In some embodiments theactive agent or modifier is provided and combined with anothercomponent, or components, as a solution. In other embodiments any of thecomponents can be provided in solid form and dissolved, or in the caseof the microparticles, suspended, in the liquid medium containinganother of the components. Further variations will be apparent to one ofskill in the art.

The microparticles are formed prior to being combined with the othercomponents of the system, and as such are present as a suspension.Nonetheless the liquid medium in which the microparticles are suspendedis at times referred to herein as a solvent. The liquid medium utilizedin the method is most often aqueous. However in some instances theliquid medium can comprise more of an organic compound, for example analcohol used as a modifier, than it does water.

Upon assembly of all components of the system, the active agent willadsorb to the surface of the microparticle. In increasingly preferredembodiments of the present invention, at least 50, 60, 70, 80, 90, 95%,or substantially all, of the active agent in the system will adsorb tothe microparticles, up to 100%. In some embodiments of the presentinvention, the accessible surface area of the microparticles with besufficient for all of the adsorbed active agent to be in direct contactwith the microparticle surface, that is, the coating is a monolayer.However it is to be understood that additional interactions can bepresent. In some instances, for example, self-association of the activeagent can also be energetically favored so that multiple layers ofactive agent coat the particle. It is not required that any of theselayers be complete or that the thickness of the coating be uniform. Twoforms of self-association can be recognized: multimerization andaggregation. Multimerization is characterized by specific intermolecularinteractions and fixed stoichiometry. Aggregation is characterized byunspecific intermolecular interactions and undefined stoichiometry. Itshould be understood that multimeric active agents can be adsorbed inthe multimeric state, or dissociated into monomers, or lower ordermultimers, and adsorbed to the surface in that state. In either caseaggregation can mediate layering of the active agent onto themicroparticle.

The loaded microparticles constitute a drug delivery composition thatcan be utilized in a variety of forms. The particles can be used aspowders, in solid dosage forms such as tablets or contained in capsules,or suspended in a liquid carrier. Generally this will require exchangeand/or removal of the liquid medium in which the loading took place.This can be accomplished by any of a variety of means including physicalmethods such as, but not limited to, sedimentation or filtration, andevaporative methods such as, but not limited to, lyophilization orspray-drying. These techniques are known to those skilled in the art. Inone embodiment of the present invention, solvent is removed byspray-drying. Methods of spray-drying diketopiperazine microparticlesare disclosed in, for example, U.S. Provisional Patent Application No.60/776,605 filed on Feb. 22, 2006, incorporated by reference herein forall it contains regarding spray-drying diketopiperazine microparticles.

If loading is not substantially complete, embodiments of the invention,using physical methods of solvent removal will typically loose theunadsorbed active agent, but for example can be useful to ensure thatcoating does not progress beyond a monolayer. Conversely, embodimentsusing evaporative drying for solvent removal can in some cases depositadditional active agent on the particle and thereby avoid its loss, butthe adsorptive interactions involved can differ from those establishedby the molecules bound in the earlier steps of the method. In otherembodiments evaporative solvent removal does not result in significantfurther deposition of active agent, including the case in whichsubstantially all of the active agent was already adsorbed to theparticle.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the present invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. While discussion may focus on aparticular mechanism it should be understood that some modifiers canhave multiple effect on the agent, or indeed on the particle surface aswell, each of which can contribute to promoting adsorption of the agentto the particle. However, those of skill in the art, in light of thepresent disclosure, will appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1

Experimental Procedure: Active Agent/FDKP Microparticle AdsorptionStudies

The active agents insulin, PTH, ghrelin and GLP-1 were either purchasedfrom American Peptide (Sunnyvale, Calif.) or AnaSpec (San Jose, Calif.),or prepared in house (MannKind Corporation, Valencia, Calif.). Aqueoussamples at varying pH and at 20° C. (unless otherwise noted) wereanalyzed. Samples were generally prepared fresh and were mixed with theparticular additive (e.g., salt, pH buffer, etc., if any), prior to theaddition of FDKP microparticles.

The association of active agent with diketopiperazine (DKP) particles insuspension was evaluated by conducting adsorption studies. Theparameters investigated in the adsorption studies explored the effectsof electrostatic interactions, hydrogen bonding, water structure,protein flexibility, and specific salt-pairing interactions on theactive agent/fumaryl diketopiperazine (FDKP) microparticle interaction.In addition, several common protein stabilizers were tested forinterference with active agent adsorption to FDKP microparticlesurfaces.

Varying conditions promoting adsorption of active agent onto thesurfaces of preformed FDKP particles were studied. A 15 mg/mL FDKPmicroparticle suspension was combined with 3× pH buffer and 3× solutionof an additive or excipient. The final solution contained a FDKPmicroparticle concentration of 5 mg/mL and a GLP-1 concentration of 0.25mg/mL (5% w/w), or a PTH concentration of 0.25 mg/mL (5% w/w), or aninsulin concentration of 0.75 mg/mL (15% w/w) or a ghrelin concentrationof 0.10 mg/mL (2% w/w). Unbound active agent in the supernatant wasfiltered off the suspension. The FDKP particles with the associatedactive agent were dissolved (reconstituted) in 100 mM ammoniumbicarbonate and filtered to separate out any aggregated active agentmolecules. The amount of active agent in both the supernatant andreconstituted fractions was quantitated by HPLC. A series of experimentswere conducted in which conditions employed included use of additivessuch as salts, osmolytes, chaotropes and kosmotropes, and alcohols. Theresults from these studies are described below.

Example 2

Effect of Chaotropes and Kosmotropes on Adsorption of Active Agent ontoFDKP Particles

Ionic species that affect the structure of water and proteins(chaotropes and kosmotropes) were studied to investigate the adsorptionof active agent onto a FDKP microparticle surface by a hydrophobicmechanism (at low pH). Loading of the active agent onto FDKP particleswas performed at 5mg/mL microparticles and a GLP-1 concentration of 0.25mg/mL (5% w/w), or a PTH concentration of 0.25 mg/mL (5% w/w), or aninsulin concentration of 0.75 mg/mL (15% w/w). The concentration of thechaotrope or kosmotrope in the samples was held constant at 100 mM andthe pH varied from 2.0 to 5.0. Chaotropes or kosmotropes were selectedfrom the following: NaSCN, CsCl, Na₂SO₄, (CH₃)₃N-HCl, Na₂NO₃, NaCitrate, and NaClO₄. The control indicates no chaotrope or kosmotropewere added.

FIGS. 1A-1C depict the loading curves for insulin, GLP-1 and PTHrespectively, onto the FDKP microparticle surface as a function of pH inthe presence of the various chaotropes or kosmotropes. At low pH (3.0)all chaotropes and kosmotropes analyzed improved the affinity of insulinfor the microparticle surface and showed significant loading compared tothe control. At pH 4, this effect was not observed (FIG. 1A). At higherpH (5.0), the chaotropes and kosmotropes interfered with the adsorptionof insulin to the microparticle surface, as compared to control, byprecipitating the insulin protein. Thus these agents promoted binding ofinsulin to the FDKP particles at lower pH, but have little or even adetrimental effect at the higher pH conditions.

GLP-1, in the presence of chaotropes and kosmotropes, showed an improvedaffinity for the FDKP microparticles at pH 2.0-4.0 with a greater effectat lower pH (FIG. 1B). Similar observations were disclosed in U.S.Provisional Application Ser. No. 60/744,882. There it was noted, thatapproximately 0.02-0.04 mg/mL of the GLP-1 peptide (which corresponds tomass ratios of 0.004 to 0.008) was detected in the reconstitutedmicroparticle-free control samples in the presence of NaSCN, NaClO₄,Na₂SO₄, NaNO₃ and Na citrate, indicating that a small proportion of theGLP-1 precipitated rather than adsorbing to the particle.

The affinity of PTH for the FDKP microparticle surface was greater at pHof 4.0 to about 4.5 in the presence of strong chaotropes NaSCN andNaClO₄ (FIG. 1C).

The data supports that chaotropic and kosmotropic agents play a role inpromoting adsorption of the active agent to FDKP microparticle surfaces,most notably at low pH. Since these modifiers have a greater effect atlow pH, where the microparticle surface is less ionic, it is likely thatadsorption results from a hydrophobic mechanism. The decrease inadsorption observed at higher pH may result from the more highly chargedsurface of the particle in combination with effects chaotropic andkosmotropic agents have on increasing the hydrophobicity of the activeagents. Additionally, as ionic species, these agents may compete withthe active agent for binding to the microparticle, or disrupt theelectrostatic interactions between the active agent and themicroparticle. Finally it is also noted that Debye shielding cancontribute to the decrease in adsorption to the more highly chargedsurface.

Example 3

Effect of Osmolytes on Adsorption of Active Agent to FDKP Particles

To assess the importance of active agent stability on adsorption, theeffect of osmolytes on the binding of active agent to FDKP particles wasexamined by HPLC analysis. FIGS. 2A-2C show the loading curves forinsulin (FIG. 2A), GLP-1 (FIG. 2B) and ghrelin (FIG. 2C) onto FDKPparticles as a function of pH in the presence of common stabilizers(osmolytes). Loading of the active agent onto FDKP microparticles wasperformed at 5 mg/mL of microparticles and an insulin concentration of0.75 mg/mL (15% w/w), or a GLP-1 concentration of 0.25 mg/mL (5% w/w) ora ghrelin concentration of 0.10 mg/mL (2% w/w). The concentration of theosmolyte (stabilizer) in the samples was held constant at 100 mM and thepH varied from about 2.0 to about 5.0. The osmolytes were selected fromhexylene-glycol (Hex-Gly), trehalose, glycine, PEG, TMAO, mannitol andproline; the control indicates no osmolyte.

Of the active agents studied, insulin showed significantly improvedaffinity for the FDKP particle surface in the presence of osmolytes(PEG, glycine, trehalose, mannitol and Hex-Gly) over a pH range of 3.0to 5.0 (FIG. 2A). Of the osmolytes studied, PEG and proline improved theaffinity for adsorption of the GLP-1 onto FDKP particle surface over apH range from 2.0 to 4.0. The osmolyte TMAO was more effective than PEGor proline at binding GLP-1 onto the FDKP microparticle surface at lowpH (2.0) but was modestly detrimental at pH 3.0 and above (FIG. 2B).Ghrelin however, showed greater affinity for the microparticle surfacein the presence of 100 mM mannitol, PEG, glycine, Hex-Gly, and trehalosewhen compared to the control over the pH range of about 4.0 to 5.0 (FIG.2C).

These loading curves suggested that osmolytes are capable of enhancingthe adsorption of the active agent to FDKP microparticle surface. It islikely that this effect resulted from the modifiers ability to stabilizethe active agent, which enabled adsorption to be more energeticallyfavorable.

Example 4

Effect of Alcohols on Affinity of Active Agent to FDKP Particles

In assessing the effect of modifiers on the active agent that allows foradsorption to the microparticle surface by a hydrophobic mechanism, theeffect of alcohols were examined. Alcohols known to induce helicalconformation in unstructured peptides and proteins by increasinghydrogen-bonding strength were evaluated to determine the role thathelical confirmation plays in adsorption of active agent to FDKPparticles surface. Active agents such as GLP-1 and ghrelin wereanalyzed. Loading of the active agent on FDKP particles was performed at5 mg/mL of microparticles and a GLP-1 concentration of 0.25 mg/mL (5%w/w) or a ghrelin concentration of 0.10 mg/mL (2% w/w). The effect ofeach alcohol was observed over a pH range of 2.0 to 5.0. The alcoholsused were trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP). Eachalcohol was evaluated at varying concentrations which include 5%, 10%,15%, or 20% v/v.

FIGS. 3A-3D show the loading curves for active agent onto FDKPmicroparticles as a function of pH for each alcohol and each activeagent. At pH 2.0-4.0, ghrelin showed greatly improved affinity for themicroparticle surface in the presence of HFIP and TFE at allconcentrations tested (5%, 10%, 15% and 20%), as demonstrated by themass ratio of ghrelin to FDKP particles (FIGS. 3A-3B).

At pH 2.0-5.0, GLP-1 showed improved affinity for the microparticlesurface in the presence of HFIP and TFE at the concentrations shown (5%and 10%) (FIGS. 3C-3D). The effect of TFE was less pronounced, and atthe lower pHs tested was detrimental. It was noted that a substantialamount of GLP-1 peptide (0.13-0.19 mg/mL, which corresponds to massratios of 0.026 to 0.038) was detected in the reconstitutedmicroparticle-free control samples in the presence of 10% HFIP and TFEat pH 4.0, indicating that some of the GLP-1 had precipitated. However,at lower pH (2.0-3.0), the amount of GLP-1 peptide detected in thereconstitued microparticle-free control in the presence of 10% HFIP orTFE was significantly decreased. At pH 3.0, GLP-1 peptide at 0 to 0.02mg/mL, (which corresponding to a mass ratio of 0 to 0.004) was detected,whereas no GLP-1 was detected for the control samples at pH 2.0. Themass ratios in FIGS. 3C-D reflect both adsorbed and precipitated activeagent although precipitation is an increasingly minor component as thepH decreased toward 3.0.

The data indicated that alcohols are able to improve the adsorption ofthe active agent onto FDKP microparticles. This increase in adsorptionlikely resulted from enhanced hydrophobic interactions between theactive agent and surface of the microparticle in the presence ofalcohols.

Example 5

Effect of Salt on Adsorption of Active Agent to FDKP Particles

To further address the hydrophobic mechanism of binding, the effects ofsalt on adsorption of active agent to FDKP microparticles were observedby HPLC analysis.

Loading of the active agent onto FDKP microparticles was performed at 5mg/mL of microparticles and an insulin concentration of 0.75 mg/mL (15%w/w), or a GLP-1 concentration of 0.25 mg/mL (5% w/w) or a PTHconcentration of 0.25 mg/mL (5% w/w) in the presence of 0, 25, 50, 100,250, and 500 mM NaCl (FIGS. 4A-4C). Loading of PTH onto FDKP particleswas also assessed at 1000 mM NaCl. The amount of active agent detectedin reconstituted microparticle-free control samples as a function of pHand NaCl concentration was assessed. The pH was controlled with a 20 mMpotassium phosphate/20 mM potassium acetate mixture.

As observed in FIG. 4A, increased binding (adsorption) of insulin ontoFDKP particles was evident at high salt concentrations of 100-500 mM atpH from about 2.5 to about 3.5. At a pH from about 4.0 to about 5.0, forall salt concentrations tested, a reduction in the adsorption of insulinto the FDKP particle was observed.

At a pH from about 2.0 to about 3.5 enhanced binding (adsorption) ofGLP-1 to FDKP particles was evident at all the salt concentrationstested (FIG. 4B). At pH 4.0 and above, a reduction in binding was alsonoted.

Similar studies using PTH as the active agent showed enhanced binding ofPTH to the FDKP particles at high salt concentrations of 250 to 1000 mMat pH from about 2.0 to about 3.5 (FIG. 4C). At pH from about 3.5 toabout 5.0 binding of PTH to the microparticle decreased in the presenceof salt.

At low pH, where adsorption is not favorable, the addition of salt wasable to modify the chemical potential of the active agent so as toincrease its affinity for the microparticle surface. Such enhancement ofbinding likely resulted from a hydrophobic mechanism. Furthermore, thedata indicated that as the pH was raised, adsorption decreased withincreased salt concentration. As the microparticle surface became morecharged with increasing pH, the hypothesized hydrophobic mechanism canbe expected to be less effective at promoting the adsorption of theactive agent. This reduction may also have resulted from salt competingfor the binding sites on the surface of the microparticle. It is notedthat Debye shielding may also contribute to the reduced adsorptionobserved

The data also showed that salt is capable of altering the structure ofactive agents. For example, circular dichroism measurements with PTHshowed that as the salt concentration increased the secondary structureof the peptide adopted a more helical conformation (FIG. 4D). Thissuggests that change in the structure of PTH may promote its binding tothe microparticle surface at low pH.

In an aqueous solution, the presence of salt was also shown to partitionthe dye Texas Red onto the surface of the microparticle.

Example 6

Effects on Cyclosporin A Adsorption to FDKP Particles

The effects on the adsorption of small hydrophobic molecules onto FDKPparticles was investigated both in vitro and in vivo using cyclosporin Aas the active agent. Adsorption was promoted by altering the solubilityof the active agent.

Cyclosporin A, a lipophilic cyclic polypeptide, was studied in order toshow how a hydrophobic molecule can be made to adsorb to microparticles.In addition, the size of cyclosporin A (1202.61 MW) was utilized todemonstrate the loading capacities of microparticles for smallercompounds.

To accomplish loading, a solvent/anti-solvent method was employed. Thebasic principle of this methodology is to dissolve the compound in asolvent (methanol) and then use anti-solvent (water) to drive thecompound out of solution and onto the surface of the microparticles.Utilizing this solvent/anti-solvent approach, cyclosporin A wassuccessfully loaded onto the surface of microparticles.

In a preliminary experiment to determine a solubility profile,cyclosporin A was dissolved to 10 mg/mL in methanol and its solubilityat 1 mg/mL with varying concentrations of anti-solvent (10-90% H₂O in10% increments) was analyzed by HPLC. The cyclosporin A peak areas werecompared against the sample containing methanol alone, to determine thepercent loss to precipitation. It was observed that solubility waslargely retained below 60% H₂O. At 70% H₂O, a significant majority ofthe agent was insoluble and at 80-90% H₂O less than 5% solubilityremained.

To assess particle loading, FDKP microparticles were suspended inmethanol solutions of cyclosporin A. Water was then added in a stepwisefashion to final concentrations of 60, 80, and 90%. Half of the samplewas pelleted and the other half lyophilized. Each half was thenredissolved such that the final percentages were 20% FDKPmicroparticles/cyclosporin A, 20% 0.5 M ammonium bicarbonate (AmBicarb),and 60% methanol (the concentrations necessary for the dissolution ofboth microparticle and cyclosporin A). The cyclosporin A content of eachwas analyzed by HPLC and compared to determine the proportion that hadbecome adsorbed to the particle. The results are presented in FIG. 5A.At 60% H₂O it was observed that about 20% of the cyclosporin A had boundto the particle. At 80% and 90% H₂O the loads were about 90% and 95%,respectively, indicating the strong binding of cyclosporin A to FDKPmicroparticles.

The loading capacity of the microparticles for cyclosporin A wasanalyzed at the 90% anti-solvent level by varying the input ofcyclosporin A so that the final content of the recovered solids would befrom 2% to 20%, assuming all of the cyclosporin A became adsorbed. Itwas observed that as the input increased over this range the percent ofavailable cyclosporin A bound to the microparticle increased from 50% to95% of the input (FIG. 5B). It is to be noted that, taking into accountthat the solubility of cyclosporin A is 0.05 mg/mL at 90% H₂O, theseresults indicated that substantially all of the insoluble cyclosporin Abecame adsorbed to the particles rather than precipitating out.

Example 7

Pulmonary Insufflation of Cyclosporin A/DKP Particles

To examine the pharmacokinetics of cyclosporin A/FDKP microparticles,plasma concentrations of cyclosporin A were evaluated in female SpragueDawley rats administered various formulations of cyclosporin A/FDKPmicroparticles via pulmonary insufflation or intravenous injection.These studies were conducted using cyclosporin A/FDKP microparticlesmade at 90% anti-solvent and a theoretical maximum mass ratio of 0.05,0.10 or 0.20 as described in the example above. These are referred to asthe 5%, 10% and 20% loads.

A single dose of 2.5 mg cyclosporin A/FDKP microparticles was deliveredto eight groups of rats via pulmonary insufflation or intravenousinjection. Blood samples were taken on the day of dosing for each groupat pre-dose (time 0), and at 5, 20, 40, 60, 240, 480 minutes and at 24hrs post dose. At each time point, approximately 100 @L whole blood wascollected from the lateral tail vein into a cryovial, inverted andstored on ice. Blood samples were centrifuged at 4000 rpm andapproximately 40 @L plasma was pipetted into 96-well plates which werestored at −80° C. until analyzed.

As shown in FIG. 6, administration of 2.5 mg FDKPmicroparticles/cyclosporin A via pulmonary insufflation resulted inmaximal serum cyclosporin levels 24 hours post dose in female SpragueDawley rats. The 10% load achieved a Cmax of 32.4 ng/mL at that timepoint. Animals administered 2.5 mg of FDKP microparticles/cyclosporin Ain 0.1 mL via intravenous injection showed minimal levels of cyclosporinout to 24 hours post dose. It was observed that FDKP microparticlelevels peaked at 20 minutes post dose and returned to baseline levels in4 hours for both the intravenous and pulmonary insufflation groups.

Overall, the data shows the bioavailability of cyclosporin A/FDKPmicroparticle. It is noted that the single peak at 240 minutes is ananomaly. For all animals treated, the pathology as determined by grossand microscopic observation was normal.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present invention. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

The terms “a” and an and the and similar referents used in the contextof describing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

The use of the term or in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

Further, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

What is claimed:
 1. A method for making a microparticle coated with anactive agent, the method comprising: i. combining (a) a microparticlecomprising crystalline fumaryl diketopiperazine, (b) an active agent,and (c) a solvent for said active agent thereby forming a suspension ofsaid microparticles in a solution of said active agent; ii. adding anactive agent modifier to said solution wherein adding said active agentmodifier causes adsorption of said active agent onto said microparticle.2. The method of claim 1, wherein said active agent modifier is a salt,surfactant, ion, osmolyte, alcohol, chaotrope, kosmotrope, acid, base,or anti-solvent
 3. The method of claim 1 further comprising removingsaid solvent subsequent to said adsorption of the active agent.
 4. Themethod of claim 2, wherein said solvent is an aqueous solvent.
 5. Themethod of claim 4 wherein said anti-solvent is an organic solvent. 6.The method of claim 2, wherein said solvent is an organic solvent. 7.The method of claim 6 wherein said anti-solvent is water.
 8. The methodof claim 2, wherein said salt is sodium chloride.
 9. The method of claim2, wherein said osmolyte is hexylene-glycol, trehalose, glycine,polyethylene glycol, trimethylamine N-oxide, mannitol, or proline. 10.The method of claim 2, wherein said alcohol is methanol, ethanol,trifluoroethanol, or hexafluoroisopropanol.
 11. The method of claim 2,wherein said chaotrope is NaSCN, (CH₃)₃N-HCl, Na₂NO₃, NaClO₄, or CsCl.12. The method of claim 2, wherein said kosmotrope is sodium citrate orsodium sulfate.
 13. The method of claim 1, wherein at least 70% of saidactive agent becomes adsorbed to said microparticle.
 14. The method ofclaim 1, wherein at least 80% of said active agent becomes adsorbed tosaid microparticle.
 15. The method of claim 1, wherein at least 90% ofsaid active agent becomes adsorbed to said microparticle.
 16. The methodof claim 1, wherein substantially all of said active agent becomesadsorbed to said microparticle.
 17. The method of claim 1, wherein saidmicroparticle is coated with a monolayer of said active agent.
 18. Themethod of claim 1, wherein adding said active agent modifier favorablymodulates one or more energetic interactions with said microparticle.19. The method of claim 18, wherein said one or more energeticallyfavorable interactions between said active agent and said microparticlecomprises a hydrophobic interaction.
 20. The method of claim 18, whereinsaid one or more energetically favorable interactions between saidactive agent and said microparticle comprises a hydrogen bondinginteraction.
 21. The method of claim 18, wherein said one or moreenergetically favorable interactions between said active agent and saidmicroparticle comprises an electrostatic interaction.
 22. The method ofclaim 1, wherein adding said active agent modifier modifies thestructure, flexibility, rigidity, solubility or stability of said activeagent.
 23. The method of claim 1, wherein said active agent is a peptideor a protein.
 24. The method of claim 1, wherein said active agent is aninsulin and an insulin analog.
 25. A method for making a microparticlecoated with an active agent that is insulin or an insulin analog, themethod comprising: (i) obtaining a crystalline fumaryl diketopiperazinemicroparticle wherein the crystalline diketopiperazine microparticledoes not comprise an active agent; (ii) forming a suspension comprisingsaid crystalline diketopiperazine microparticle and a solvent; (iii)dissolving said active agent in the fluid phase of said suspension; (iv)increasing the pH of the fluid phase to about 4 to 5; (v) adsorbing ofsaid active agent onto a surface of said crystalline diketopiperazinemicroparticle to provide a coating of said active agent on saidcrystalline diketopiperazine microparticle; and (vi) removing orexchanging said solvent after step (v).